We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.
- Authors
DiCarlo, James E; Norville, Julie E; Mali, Prashant; Rios, Xavier; Aach, John; Church, George M
- Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.
- Publication
Nucleic acids research, 2013, Vol 41, Issue 7, p4336
- ISSN
1362-4962
- Publication type
Journal Article
- DOI
10.1093/nar/gkt135